1. Field of the Invention
This invention relates to methods for using volume exclusion agents to enhance in situ hybridization between polynucleotide probes and their target polynucleotides, particularly in an automated testing environment. In one aspect, the invention specifically relates to the use of volume exclusion agents to facilitate assay and diagnostic procedures for the detection of DNA and RNA sequences, particularly human papillomavirus (HPV), Epstein Bar virus (EBV), human immunoglobulin light chain mRNA (Kappa and Lambda sequences), and Her-2/neu gene.
2. Description of Related Art
Hybridization is a general technique in which the complementary strands of deoxyribonucleic acid (hereinafter xe2x80x9cDNAxe2x80x9d) molecules, ribonucleic acid (hereinafter xe2x80x9cRNAxe2x80x9d) molecules, and combinations of DNA and RNA are separated into single strands and then allowed to renature or reanneal into base-paired double helices. At least three major classes of hybridization are conventionally known and used: solution hybridization which disrupts the individual cells and extracts the internal nucleic acids into solution prior to hybridization; filter or blot hybridization which transfers extracted DNA (or RNA) fragments from agarose gels to filters or blotters such as cellulose nitrate or nylon for subsequent hybridization with radioactive DNA or (RNA) and then detection of hybridization by radioautography or fluorography; and in situ hybridization (xe2x80x9cISHxe2x80x9d) which makes possible the detection and localization of specific nucleic acid or polynucleotide sequences directly within a structurally intact cell or cellular component where extraction of nucleic acids from the cell is undesirable. Although each of these respective hybridization techniques often employ cells, tissues, and certain reagents in common, each technique is generally viewed and accepted within this art as different and completely distinguishable from any other.
In situ hybridization is a technique which yields both molecular and morphological information about intact individual cells and cellular parts. Rather than requiring the investigator to laboriously extract DNA and/or RNA from a heterogeneous cell population, the technique permits detection of DNA and RNA in-situ within the cellular morphology and allows the investigator to identify those particular cells or cell parts which contain specific DNA or RNA sequences of interest. This technique also allows one to determine simultaneously the biochemical and/or morphological characteristics of these cells. For this reason, the in situ hybridization methodology has direct application for many areas of biomedical and clinical research including developmental biology, cell biology, genetics, clinical diagnosis, and pathological evaluation.
Despite the potential of in-situ hybridization as a molecular analytical technique, the development of effective protocols and procedures has been largely haphazard and disjointed. Since first described in 1969 by Gall et al., P.N.A.S. U.S.A., 63:378-383 (1969); Methods in Enzymol., 38:370-380 (1971), the in situ hybridization approach has been directed towards two different morphological situations: the localization of specific nucleic acid sequences of interest in the cytoplasm of a cell; and the identification of specific nucleic acids within the nucleus and/or chromosomes of a cell.
Much of the research related to hybridization between target and probe polynucleotides for assay and diagnostic purposes has been directed toward optimizing rates of hybridization. In situ hybridization is particularly problematic due to the inability of the probes to readily enter into the nucleus or cytoplasm in which their target polynucleotides are located. To solve this problem, researchers have attempted, inter alia, to reduce the size of the probe and to alter cell fixation procedures to facilitate entry of the probe into the cytoplasm or nucleus, see generally Singer, R. H., et al., xe2x80x9cOptimization of In Situ Hybridization Using Isotopic and Non-Isotopic Detection Methods,xe2x80x9d Biotechniques 4(3):230-250, 1986, and Haase, A., et al., xe2x80x9cDetection of Viral Nucleic Acids by In Situ Hybridization,xe2x80x9d Methods in Virology, Vol. VII, pp. 189-226, (1984). Amasino, R. M., xe2x80x9cAcceleration of Nucleic Acid Rate by Polyethylene Glycol,xe2x80x9d Anal. Biochem., 152:304-307 (1986). It has been reported that the effect of dextran sulfate, the most commonly used exclusion agent, was most pronounced in mixed phase hybridizations where the probes exceeded 250 nucleotides. Further, it has been reported that as the probe size decreases, so would the enhancing effect of dextran sulfate on the rate of hybridization, with no effect observed for oligonucleotides of 14 bases. Meinkoth J. and Wahl J., xe2x80x9cHybridization of Nucleic Acids Immobilized on Solid Supportsxe2x80x9d (Review), Anal. Biochem., 138:267-284 at 268 (1984). The use of volume exclusion to enhance in situ hybridization has also been reported. It was reported that an average length of 400 nucleotides is optimal for hybridization in situ in the presence of dextran sulfate. Hasse, A., supra. at 205.
Early references that disclose the use of dextran sulfate as a volume exclusion agent include Wahl, G. M., et al., xe2x80x9cEfficient transfer of large DNA fragments from agarose gels to diazobezyloxymethyl-paper and rapid hybridization using dextran sulfate,xe2x80x9d PNAS 76: 3683 (1979); and Ledermann, L. L., et al., xe2x80x9cThe rate of nucleic acid annealing to cytological preparations is increased by the presence of dextran sulfate,xe2x80x9d Anal. Biochem., 117(1): 158-163 (1981).
The in situ localization of HPV DNA using long biotinylated probes in the presence of dextran sulfate has also been reported by Beckmann, P. M., et al.; xe2x80x9cDetection and Localization of Human Papillomavirus DNA in Human Genital Condylomas by In Situ Hybridization with Biotinylated Probes,xe2x80x9d J. Med. Virol., 16:265-273 (1985); Milde K., Loning, T., xe2x80x9cDetection of Papillomavirus DNA in Oral Papillomas and Carcinomas: Application of In Situ Hybridization with Biotinylated HPV 16 probes,xe2x80x9d J. Oral Pathol., 15:292-296 (1986); and McDougall, J. K., et al., xe2x80x9cMethods for Diagnosing Papillomavirus Infection,xe2x80x9d in Papillomaviruses, Wiley, Chicester (CIBA Foundation Symposium 120), pp. 86-103 (1986).
U.S. Pat. No. 5,985,549 (Singer, et al.) demonstrates the use of a dextran sulfate hybridization buffer containing formamide (deionized); dextran sulfate (10%); human DNA or salmon sperm DNA (100 ug/ml); human tRNA (100 ug/ml); and vanadyl sulfate (10 uM) for ISH. The molecular weight of dextran sulfate was not disclosed, and it is assumed that 500,000 average molecular weight was obtained.
U.S. Pat. No. 5,750,340 (Kim, I., et al.) disclose a hybridization solution for performing ISH, the solution consisting essentially of 8-12% dextran sulfate, 10-30% formamide, and a salt. No source for the dextran sulfate, or molecular weight, is specified.
U.S. Pat. No. 5,116,727 (Brigatti) discloses hybridization buffers that contain anionic heteropolysaccharides (e.g. chondroitin A sulfate) as useful volume exclusion agents for accelerating hybridization reactions. Chondroitin A sulfate hybridization buffers were of low viscosity which was a useful property for capillary gap slides and their use in the automated processing of in situ hybridization reactions. Brigatti teaches that the low viscosity of this buffer is due to the volume exclusion agent having an anionic heteropolysaccharide structure; this was compared to anionic polysaccharides like dextran sulfate. Brigatti further discloses that anionic homopolysaccharides like dextran sulfate polymers produce buffers of substantially greater viscosity based on the their monomeric structure. This high viscosity makes such hybridization buffers non-ideal for capillary gap technology since high viscosity inhibits both probe diffusing in and out from target and during wash steps to wash away excess probe. Brigatti further teaches that increasing the concentration of dextran sulfate also increases the viscosity and thus inhibits the hybridization process.
U.S. Pat. No. 4,886,741 (Schwartz et al.) describe the use of dextran sulfate, sodium salt, for use as a volume exclusion agent for ISH. The average molecular weight is not described, but by reference to the source (Sigma Chemical, Products for Life Sciences, St. Louis, Mo.) it has an average molecular weight of 500,000. Schwartz et al. also disclose that dextran sulfate is typically used at a concentration of about 5-10% (w/v).
U.S. Pat. No. 4,302,204 (Wahl, G., et al.), describes the use of dextran sulfate polymers for hybridization buffers used for in vitro blot hybridization. In this patent however, the preferred hybridization buffer contained dextran sulfate of 500,000 MW. No examples were presented that used low molecular weight dextran sulfate nor was an in situ reaction presented or claimed.
In the field of nucleic acid hybridization, the need for rapid assay tests for the accurate and reproducible detection of nucleic acids has been a long standing problem. Any procedures that demonstrate a tendency to accelerate the typically multi-hour long processes are of value, especially for hybridization assays to be conducted by clinical laboratories.
This invention demonstrates that lower molecular weight dextran sulfate is also an effective volume exclusion agent for hybridization reactions. The hybridization buffers of this invention utilize smaller polymers of dextran sulfate, and thus are of a lower viscosity than conventional hybridization buffers that use higher molecular weight dextran sulfate polymers as volume exclusion agents. The lower viscosity of these low molecular weight dextran sulfate polymer buffers makes them useful for automated hybridization processes as in dispensing, automated flow, and mixing on slide.
The invention is directed to an improvement in automated tissue staining system having evaporation inhibitor liquid covering the polynucleotide hybridization buffer-covered tissue on a slide, wherein the improvement comprises a polynucleotide hybridization buffer for in situ hybridization comprising a low molecular weight dextran sulfate having a molecular weight range from about 8,000 to about 16,000.